Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation.

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the 'four-dimensional' protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the 'toolbox' of mass spectrometry researchers studying higher-order protein structure.

Design and Combinatorial Development of Shield-1 Peptide Mimetics Binding to Destabilized FKBP12.

On the basis of computational design, a focused one-bead one-compound library has been prepared on microparticle-encoded PEGA1900 beads consisting of small tripeptides with a triazole-capped N-terminal. The library was screened towards a double point-mutated version of the human FKBP12 protein, known as the destabilizing domain (DD). Inspired by the decoded library hits, unnatural peptide structures were screened in a novel on-bead assay, which was useful for a rapid structure evaluation prior to off-bead resynthesis. Subsequently, a series of 19 compounds were prepared and tested using a competitive fluorescence polarization assay, which led to the discovery of peptide ligands with low micromolar binding affinity towards the DD. The methodology represents a rapid approach for identification of a novel structure scaffold, where the screening and initial structure refinement was accomplished using small quantities of library building blocks.

Bifunctional Small-Molecule Ligands of K-Ras Induce Its Association with Immunophilin Proteins.

Here we report the design, synthesis, and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin, and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Ras-driven cancers.

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease.

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.

Activation of BMP Signaling by FKBP12 Ligands Synergizes with Inhibition of CXCR4 to Accelerate Wound Healing.

The combination of AMD3100 and low-dose FK506 has been shown to accelerate wound healing in vivo. Although AMD3100 is known to work by releasing hematopoietic stem cells into circulation, the mechanism of FK506 in this setting has remained unknown. In this study, we investigated the activities of FK506 in human cells and a diabetic-rat wound model using a non-immunosuppressive FK506 analog named FKVP. While FKVP was incapable of inhibiting calcineurin, wound-healing enhancement with AMD3100 was unaffected. Further study showed that both FK506 and FKVP activate BMP signaling in multiple cell types through FKBP12 antagonism. Furthermore, selective inhibition of BMP signaling abolished stem cell recruitment and wound-healing enhancement by combination treatment. These results shed new light on the mechanism of action of FK506 in acceleration of wound healing, and raise the possibility that less toxic FKBP ligands such as FKVP can replace FK506 for the treatment of chronic wounds.

Computational design of chemogenetic and optogenetic split proteins.

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites.

The dTAG system for immediate and target-specific protein degradation.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

A calcineurin antifungal strategy with analogs of FK506.

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.

A method to rapidly create protein aggregates in living cells.

The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.

II. Dissociation free energies in drug-receptor systems via nonequilibrium alchemical simulations: application to the FK506-related immunophilin ligands.

The recently proposed fast switching double annihilation (FS-DAM) [Cardelli et al., J. Chem. Theory Comput., 2015, 11, 423] is aimed at computing the absolute standard dissociation free energies for the chemical equilibrium RL ⇌ R + L occurring in solution through molecular dynamics (MD) simulations at the atomistic level. The technique is based on the production of fast nonequilibrium annihilation trajectories of one of the species (the ligand) in the solvated RL complex and in the bulk solvent. As detailed in the companion theoretical paper, the free energies of these two nonequilibrium annihilation processes are recovered by using an unbiased unidirectional estimate derived from the Crooks theorem exploiting the inherent Gaussian nature of the annihilation work. The FS-DAM technique was successfully applied to the evaluation of the dissociation free energy of the complexes of Zn(ii) cations with an inhibitor of the Tumor Necrosis Factor α converting enzyme. Here we apply the technique to a real drug-receptor system, by satisfactorily reproducing the experimental dissociation free energies of FK506-related bulky ligands towards the native FKBP12 enzyme and by predicting the dissociation constants for the same ligands towards the mutant I56D. The effect of such mutations on the binding affinity of FK506-related ligands is relevant for assessing the thermodynamic forces regulating molecular recognition in FKBP12 inhibition.

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

UNLABELLED: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms.

The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.

Protein-ligand docking using hamiltonian replica exchange simulations with soft core potentials.

Molecular dynamics (MD) simulations in explicit solvent allow studying receptor-ligand binding processes including full flexibility of the binding partners and an explicit inclusion of solvation effects. However, in MD simulations, the search for an optimal ligand-receptor complex geometry is frequently trapped in locally stable non-native binding geometries. A Hamiltonian replica-exchange (H-REMD)-based protocol has been designed to enhance the sampling of putative ligand-receptor complexes. It is based on softening nonbonded ligand-receptor interactions along the replicas and one reference replica under the control of the original force field. The efficiency of the method has been evaluated on two receptor-ligand systems and one protein-peptide complex. Starting from misplaced initial docking geometries, the H-REMD method reached in each case the known binding geometry significantly faster than a standard MD simulation. The approach could also be useful to identify and evaluate alternative binding geometries in a given binding region with small relative differences in binding free energy.

Classical force field parameters for two high-affinity ligands of FKBP12.

FKBP12 is an important target in the treatment of transplant rejection and is also a promising target for cancer and neurodegenerative diseases. We determined for two ligands of nanomolar affinity the set of parameters in the CHARMM force field. The fitting procedure was based on reproducing the quantum chemistry data (distances, angles, and energies). Since the dynamical behavior of such ligands strongly depends on the dihedral angles, care was taken to derive the corresponding parameters. Moreover, since each of the central core region of these two ligands is similar to other known ligands or drugs of other proteins, part at least of these parameters could also be useful for these other ligands.

Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic ß islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Šresolution from P21 and P3121 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3121 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P21 crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Construction of unnatural heterodimeric receptors based on IL-2 and IL-6 receptor subunits.

Cells have various receptors on their surface for responding to extracellular signals that involve intercellular communication. Although the mechanism of signal transduction by such wild-type receptors has been studied intensively, there has been minimal effort in investigating whether such receptors could signal when unnaturally coupled. In this study, we investigated whether unnatural receptor pairs comprising interleukin-2 (IL-2) and interleukin-6 (IL-6) receptor subunits could transduce a signal through forced dimerization. We replaced the extracellular domain of IL-2R and IL-6R signaling subunits (IL-2Rß, IL-2Rγ, and gp130) with the FK506-binding protein (FKBP) or the FKBP12-rapamycin binding (FRB) domain, the protein pair known to be heterodimerized by rapamycin. When expressed in a hematopoietic cell line, unnatural heterodimers (IL-2Rß-gp130 and IL-2Rγ-gp130 pairs) successfully transduced a signal. While the IL-2Rγ-gp130 pair maximally mimicked gp130 signaling, the IL-2Rß-gp130 pair gave weaker gp130 signaling and no significant induction of IL-2Rß signaling, indicating a high potential of the IL-2Rγ chain in terms of activating the coupled partners. This is the first report demonstrating that heterodimeric combinations of IL-2R and IL-6R subunits are functional for signaling. Further extension of this approach may attain a creative design of artificial receptor pairs that have distinct signaling properties when compared with natural receptors.

Peptidyl-prolyl isomerase activity of FK506 binding protein 12 prevents tau peptide from aggregating.

The Alzheimer's disease-related protein, tau, aggregates into neurofibrillary tangles when it is hyperphosphorylated. The amino acid sequence included in the third repeat (R3) of the microtubule-binding region is suspected to be the main factor for tau aggregation. Here, we synthesized a 31-residue oligopeptide, corresponding to the R3 region, and characterized its aggregation propensity under various conditions. This peptide aggregated even in the absence of an aggregation-inducing molecule at a low salt concentration, while it did not form any aggregates at a high salt concentration. This suggests that hydrophilic interactions are the main cause of aggregation. We then investigated the function of FK506-binding protein (FKBP) 12, which is known to accumulate in neurofibrillary tangles in vivo, on aggregation of the R3 peptide and found that FKBP12 completely prevented the peptide from aggregating at a concentration ratio of 1 : 4 (peptide:FKBP12). FKBP12 also restored the oligomer of the peptide to its monomeric status. Mutational studies on the catalytic center of FKBP12 indicated that peptidyl-prolyl isomerase activity of FKBP12 was essential for prevention of aggregation. Assuming that the propensity of aggregation of the peptide is different in each cis-/trans-isomer, we propose that the aggregation behavior of the R3 peptide can be theoretically described with a simple kinetic scheme, in which only the cis-isomer can aggregate and FKBP12 catalyzes isomerization of the peptide in both the monomeric and aggregative states.

A rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome.

Sjögren's syndrome (SjS) is a chronic autoimmune disease characterized initially by lymphocytic infiltration and destruction of exocrine glands, followed by systemic organ damage and B-cell lymphoma. Conventional treatment is based on management of symptoms and there is a shortage of therapies that address the underlying causes of inflammation at source exocrine tissue. The aim of this study was to test a novel protein polymer-based platform consisting of diblock copolymers composed from Elastin-like Polypeptides (ELPs) fused with FKBP12, to deliver a potent immunosuppressant with dose-limiting toxicity, rapamycin (Rapa) also known as Sirolimus, and evaluate its effects on the inflamed lacrimal gland (LG) of non-obese diabetic mouse (NOD), a classic mouse model of SjS. Both soluble and diblock copolymer ELPs were fused to FKBP12 and characterized with respect to purity, hydrodynamic radii, drug entrapment and release. Both formulations showed successful association with Rapa; however, the nanoparticle formulation, FSI, released drug with nearly a 5 fold longer terminal half-life of 62.5h. The strong interaction of FSI nanoparticles with Rapa was confirmed in vivo by a shift in the monoexponential pharmacokinetic profile for free drug to a biexponential profile for the nanoparticle formulation. When acutely administered by injection into NOD mice via the tail vein, this FSI formulation significantly suppressed lymphocytic infiltration in the LG relative to the control group while reducing toxicity. There was also a significant effect on inflammatory and mammalian target of Rapamycin (mTOR) pathway genes in the LG and surprisingly, our nanoparticle formulation was significantly better at decreasing a proposed tear biomarker of SjS, cathepsin S (CATS) compared to free drug. These findings suggest that FSI is a promising tool for delivering Rapa for treatment of SjS in a murine model and may be further explored to meet the unmet medical challenge of SjS.